Construction Of GDB124 Interference Lentiviral Vectors And Stable Knock-Down Cell Line

Goat β-defensin 124(gDB124)is highly expressed in the epididymal caput of the Taihang black goat and is located in ciliary layer in epididymal head epithelial cells and luminal fluid,according to previous results by high-throughput transcriptome sequencing of the epididymal caput and testis of Taihang black goats it is speculated that it may be related to sperm maturation and capacitation.However,this the mechanism is still unclear yet.Therefore,this study was to establish gDB124 stable knock-down cell line in order to investage the effect gDB124 knock-down on immature spermatozoa of Taihang black goat,and to explore the downstream signal pathways after knock-down gDB124.In this study,epididymal head tissue of Taihang black goats was collected and digested by trypsin for establishing epididymal caput cell line of Taihang black goat.Optimum MOI values of epididymal caput cells in Taihang black goats were screened by LV10-NC lentivirus negative control vector.Three lentiviral vectors were transfected to epididymal caput cells of Taihang black goats at optimal MOI value,the examination of mRNA expression of gDB124 by fluorescence quantitative and fluorescence detection for screening of the most efficient lentiviral vectors.Epididymal caput cells of Taihang black goats was treated with final concentrations of 0,1,2.5,5,7.5,10μg/mL puromycin,the minimum lethal concentration of puromycin was confirmed based on the number of viable cells after 72 hours.The positive cells of knock-down gDB124 were screened by puromycin,after conducting several subcultures,cryopreservation,and resuscitation,a stable knock-down gDB124 cell line was finally established.The results are as follows:(1)The epididymal caput cell line of Taihang black goats was successfully established.The growth curve of the goat epididymal caput cells showed an "S" pattern as a whole,which was conformed to the cell growth rule,and they were able to stably passage and grow well.(2)Three knock-down gDB124 lentiviral vectors were:LV10-gDB 124-51,LV10-gDB124-104,and LV10-gDB124-161.The shRNA sequences were verified by sequencing.The titers were 1 × 108 TU/mL,3 × 108 TU/mL and 1 × 108 TU/mL by packing of lentivirus,respectively.(3)The optimal MOI number for LV10-NC lentiviral vector infection in epididymal caput cells of Taihang black goats was 1.The effective interference vectors screened by QRT-PCR is LV10-gDB 124-51 and LV10-gDB124-161 with equal cotransfection(P<0.0005),the silencing rate was over 50%.(4)The final concentration of puromycin in the epididymal head cells of Taihang black goat was 1 μg/mL.(5)The positive cells that could stably passage and express fluorescence were obtained,after LV0-gDB 124-51 and LV10-gDB124-161 lenti viruses invaded epididymal caput cells of Taihang black goats.In conclusion,this study successfully established the epididymal caput cell line of Taihang black goat,and constructed three lentiviral vectors that could effectively silence gDB124,and successfully established a stable knock-down gDB124 cell line.